Protein biosynthesis involves the participation of a large number of species. Of particular interest are those protein factors which are involved in the itiation of translation. Initiation factor 3 (IF3) is crucial for this process by ensuring that a pool of free 30S ribosomal subunits is available and by stimulating the func- tional interaction of mRNA with the 30S subunit. Several questions remain outstanding with regard to the biosynthesis of IF3 and its functional interaction with the ribosome. The gene encoding IF3, infC, has been cloned in this laboratory and several promoters which control the expression of the gene have been identified. Promoter fusion and S1 nuclease mapping studies will be carried out in order to analyze the activity and regulation of transcription which is initiated from each of these promoters. These will include an investigation of the growth rate regulation of infC expression and the possible involvement of stringent control. Little is currently known regarding the structure-function relationships of IF3. Mutant forms of IF3 will be produced by both deletion and site-specific mutagenesis. The biologicaL activities of the mutant proteins will be determined in a variety of functional assays. This should provide information on the composition of the active site of IF3. Previous studies in this laboratory have shown that IF3 and its gene have been evolutionarily conserved. The infC-like genes and IF3 from several organisms other than E. coli will be isolated sequenced and functionally characterized. This data should facilitate identification of those domains of IF3 which have been conserved and are presumably required for function. The protein components of the ribosome binding site for IF3 have been identified but this has not been accomplished for the 16S RNA portion of the site. This will be analyze by the construction of 30S ribosomal subunits with site-specific mutations in the 16S RNA. The ability of IF3 to bind to these mutant subunits will be assessed. The proposed experiments will clarify aspects of the regulation of IF3 biosynthesis provide a more comprehensive description of the structure-function relationships of and reveal the involvement of specific 16S RNA sequences in IF3 binding. Since IF3 plays central role in the initiation of protein synthesis, these studies will, in turn allow for a more complete understanding of the molecular mechanisms of this critical step in gene expression.